Nerve Growth Factor Receptor Gene Research Articles

Endurance Training and Exogenous Adenosine Infusion Mitigate Hippocampal Inflammation and Cell Death in a Rat Model of Cerebral Ischemia/Reperfusion Injury: A Randomized Controlled Trial

Background: Cerebral ischemia can cause irreversible structural and functional damages to the brain, especially to the hippocampus. Preconditioning with endurance training and endogenous adenosine infusion may reduce ischemia-associated damages. Objectives: This study aimed to evaluate the effect of preconditioning with endurance training and endogenous adenosine infusion on cell death in the hippocampal CA1 region following ischemia/reperfusion injuries in a rat model. Methods: Male Wistar rats were divided into five groups: (1) control (n = 8); (2) ischemia (n = 12); (3) endurance training + ischemia (n = 12); (4) adenosine infusion + ischemia (n = 12); and (5) endurance training + adenosine infusion + ischemia (n = 12). The rats in the training groups ran on a treadmill five days per week for eight weeks. In the adenosine infusion groups, the rats were injected 0.1 mg/mL/kg of adenosine intraperitoneally. Also, in the ischemic groups, both common carotid arteries were clamped for 45 minutes. Cresyl violet staining and real-time polymerase chain reaction (PCR) assay were used to evaluate cell death and cytokine gene expression, respectively. Results: Based on the present results, treatments, including endurance training + ischemia, adenosine infusion + ischemia, and endurance training + adenosine infusion + ischemia reduced the level of interleukin-6 (IL-6) and glutamate gene expression, respectively, compared to the group of ischemia only. In contrast, the expression of nerve growth factor (NGF) and adenosine receptor (A2A) genes increased by seven, four, and two folds in the endurance training + ischemia, adenosine infusion + ischemia, and endurance training + adenosine infusion + ischemia groups, respectively, compared to the group of ischemia only. Conclusions: Endurance training on a treadmill and exogenous adenosine infusion synergistically diminished cell death and reduced the expression of pro-inflammatory cytokines, while promoting the neurotrophic factor expression. When endurance training and adenosine infusion were used as stimulants before the induction of cerebral ischemia, they significantly reduced cell death.

Associations between polymorphisms of SLC22A7, NGFR, ARNTL and PPP2R2B genes and Milk production traits in Chinese Holstein

BackgroundOur preliminary work confirmed that, SLC22A7 (solute carrier family 22 member 7), NGFR (nerve growth factor receptor), ARNTL (aryl hydrocarbon receptor nuclear translocator like) and PPP2R2B (protein phosphatase 2 regulatory subunit Bβ) genes were differentially expressed in dairy cows during different stages of lactation, and involved in the lipid metabolism through insulin, PI3K-Akt, MAPK, AMPK, mTOR, and PPAR signaling pathways, so we considered these four genes as the candidates affecting milk production traits. In this study, we detected polymorphisms of the four genes and verified their genetic effects on milk yield and composition traits in a Chinese Holstein cow population.ResultsBy resequencing the whole coding region and part of the flanking region of SLC22A7, NGFR, ARNTL and PPP2R2B, we totally found 20 SNPs, of which five were located in SLC22A7, eight in NGFR, three in ARNTL, and four in PPP2R2B. Using Haploview4.2, we found three haplotype blocks including five SNPs in SLC22A7, eight in NGFR and three in ARNTL. Single-SNP association analysis showed that 19 out of 20 SNPs were significantly associated with at least one of milk yield, fat yield, fat percentage, protein yield or protein percentage in the first and second lactations (P < 0.05). Haplotype-based association analysis showed that the three haplotypes were significantly associated with at least one of milk yield, fat yield, fat percentage, protein yield or protein percentage (P < 0.05). Further, we used SOPMA software to predict a SNP, 19:g.37095131C > T in NGFR, changed the structure of NGFR protein. In addition, we used Jaspar software to found that four SNPs, 19:g.37113872C > G,19:g.37113157C > T, and 19:g.37112276C > T in NGFR and 15:g.39320936A > G in ARNTL, could change the transcription factor binding sites and might affect the expression of the corresponding genes. These five SNPs might be the potential functional mutations for milk production traits in dairy cattle.ConclusionsIn summary, we proved that SLC22A7, NGFR, ARNTL and PPP2R2B have significant genetic effects on milk production traits. The valuable SNPs can be used as candidate genetic markers for genomic selection of dairy cattle, and the effects of these SNPs on other traits need to be further verified.

NGF Signaling Interacts With the Hippo/YAP Pathway to Regulate Cervical Cancer Progression.

Nerve growth factor (NGF) is increasingly implicated in cervical cancer progression, but its mechanism in cervical cancer is unclear. Here, studies demonstrate that NGF inhibits the Hippo signaling pathway and activates Yes-associated protein (YAP) to induce cervical cancer cell proliferation and migration. Our results suggested that stimulation of NGF promoted cell growth and migration and activated YAP in HeLa and C-33A cell lines. The expression of YAP target genes (CTGF and ANKRD1) was upregulated after NGF treatment. The NGF inhibitor Ro 08-2750 and siRNA-mediated NGF receptor gene silencing suppressed HeLa and C-33A cells proliferation and migration, activated large suppressor kinase 1 (LATS1) kinase activity, and suppressed YAP function. In addition, the expression of YAP target genes (CTGF and ANKRD1) was suppressed by Ro 08-2750 treatment in HeLa and C-33A cells. Interestingly, proliferation was significantly higher in NGF-treated cells than in control cells, and this effect was completely reversed by the YAP small molecule inhibitor-verteporfin. Furthermore, the mouse xenograft model shows that NGF regulates YAP oncogenic activity in vivo. Mechanistically, NGF stimulation inactivates LATS1 and activates YAP, and NGF inhibition was found to induce large suppressor kinase 1 (LATS1) phosphorylation. Taken together, these data provide the first direct evidence of crosstalk between the NGF signaling and Hippo cancer pathways, an interaction that affects cervical cancer progression. Our study indicates that combined targeting of the NGF signaling and the Hippo pathway represents a novel therapeutic strategy for treatment of cervical cancer.

Metformin inhibits proliferation of oral squamous cell carcinoma cells by suppressing proteolysis of nerve growth factor receptor

ObjectiveWe aimed to explore the effects of metformin on oral squamous cell carcinoma (OSCC) cell proliferation and the associated molecular mechanisms. MethodsWe established an OSCC model in SCC15 cells overexpressing nerve growth factor receptor (NGFR) or the N-terminal region (aa 1–250; NGFR-N), and assessed cell proliferation by CCK-8 assay, colony formation assay, and cell cycle analysis. Levels of NGFR and related genes and proteins were detected by qPCR and western blotting, and NGFR and NGFR-N affinity for p53 was assessed by immunoprecipitation assay. Additionally, the effects of NGFR and NGFR-N on p53 binding with its downstream gene promoters were analyzed by chromatin immunoprecipitation. ResultsMetformin inhibited OSCC cell proliferation and blocked NGFR proteolysis, thereby reducing the generation of its intracellular domain and NGFR-N. Moreover, compared with NGFR, NGFR-N showed higher affinity for p53 and more strongly inactivated p53 to promote cell proliferation. Furthermore, upregulation of NGFR-N downregulated levels of p53-specific downstream transcripts and proteins, whereas these levels were significantly upregulated in metformin-treated cells overexpressing NGFR. ConclusionsThese results showed that metformin inhibited cell proliferation by suppressing NGFR proteolysis, thereby promoting its antitumor effect in OSCC and offering novel insight into a role for metformin in OSCC treatment.

Role of TrkA signaling during tadpole tail regeneration and early embryonic development in Xenopus laevis.

Neurotrophic signaling regulates neural cell behaviors in development and physiology, although its role in regeneration has not been fully investigated. Here, we examined the role of neurotrophic signaling in Xenopus laevis tadpole tail regeneration. After the tadpole tails were amputated, the expression of neurotrophin ligand family genes, especially ngf and bdnf, was up-regulated as regeneration proceeded. Moreover, notochordal expression of the NGF receptor gene TrkA, but not that of other neurotrophin receptor genes TrkB and TrkC, became prominent in the regeneration bud, a structure arising from the tail stump after tail amputation. The regenerated tail length was significantly shortened by the pan-Trk inhibitor K252a or the TrkA inhibitor GW-441756, but not by the TrkB inhibitor ANA-12, suggesting that TrkA signaling is involved in elongation of regenerating tails. Furthermore, during Xenopus laevis embryonic development, TrkA expression was detected in the dorsal mesoderm at the gastrula stage and in the notochord at the neurula stage, and its knockdown led to gastrulation defects with subsequent shortening of the body axis length. These results suggest that Xenopus laevis TrkA signaling, which can act in the mesoderm/notochord, plays a key role in body axis elongation during embryogenesis as well as tail elongation during tadpole regeneration.

No Association of Polymorphisms in Nav1.7 or Nerve Growth Factor Receptor Genes with Trigeminal Neuralgia.

Trigeminal neuralgia is defined as a sudden severe shock-like pain within the distribution of the trigeminal nerve. Pain is a subjective experience that is influenced by gender, culture, environment, psychological traits, and genes. Sodium channels and nerve growth factor play important roles in the transmission of nociceptive signals and pain. The aim of this study was to investigate the occurrence of Nav1.7 sodium channel and nerve growth factor receptor TrkA gene polymorphisms (SCN9A/rs6746030 and NTRK1/rs633, respectively) in trigeminal neuralgia patients. Ninety-six subjects from pain specialty centers in the southeastern region of Brazil were divided into 2 groups: 48 with classical trigeminal neuralgia diagnosis and 48 controls. Pain was evaluated using the visual analog scale and multidimensional McGill Pain Questionnaire. Genomic DNA was obtained from oral swabs in all individuals and was analyzed by real-time polymerase chain reaction. No association was observed between evaluated polymorphisms and trigeminal neuralgia. For allele analyses, patients and controls had similar frequencies for both genes. Genotype distribution or allele frequencies of polymorphisms analyzed here did not correlate to pain scores. Although no association of evaluated polymorphisms and trigeminal neuralgia diagnosis or pain severity was observed, our data do not exclude the possibility that other genotypes affecting the expression of Nav1.7 or TrkA are associated with the disease. Further studies should investigate distinct genetic polymorphisms and epigenetic factors that may be important in expression of these molecules.

Overexpression of hsa-miR-939 follows by NGFR down-regulation and apoptosis reduction.

Neurotrophin receptors play a crucial role in neuronal survival, differentiation and regeneration. Nerve growth factor receptor (NGFR) or P75NTR is a neurotrophin receptor that is involved in many pathological conditions including cancers. Genetic factors that are involved in regulation of neurotrophin receptors are under intense investigation. MiRNAs are novel regulators of signalling pathways that are candidates for regulation of neurotrophin receptors. Computational programs predicted that NGFR gene is a bona fide target for hsa-miR- 939. RT-qPCR, Western analysis and dual luciferase assay evidences indicated that NGFR transcript is targeted by hsa-miR-939. Also, hsa-miR-939 overexpression brought about down-regulation of NGFR expression in U87 cell line, followed by cell death rate reduction, detected by flow cytometry. Taken together, here for the first time, hsa-miR-939 is introduced as a novel key regulator of NGFR expression and its involvement in cell death/survival processes is suggested.

Allogeneic Genetically Modified T Cells (HSV-TK) As Adjunctive Treatment in Haploidentical Hematopoietic Stem-Cell Transplantation (haplo-HSCT) of Adult Patients with High-Risk Hematological Malignancies: A Pair-Matched Analysis from the Acute Leukemia Working Party of EBMT

Introduction. Current approaches to haplo-HSCT rely either on T-cell depletion to overcome the HLA disparity or on the administration of the lymphotoxic agent cyclophosphamide several days after stem cell infusion, with the goal of selectively depleting activated alloreactive lymphocytes in vivo. In both approaches, haplo-HSCT can be associated with prolonged immunodeficiency post-transplantation. Thus, effective approaches to hastening immune reconstitution following transplantation are needed. Zalmoxis¨ is an Advanced Therapy Medicinal Product based on somatic T-cells genetically modified to express the Herpes Simplex Thymidine Kinase (HSV-TK) suicide gene and a truncated form of the human Low Affinity Nerve Growth Factor Receptor (ΔLNGFR) genes (for identification of transduced cells). The expression of the HSV-TK gene, as a suicide gene allows the selective killing of dividing cells upon administration of the pro-drug ganciclovir (GCV). If GvHD occurs, ganciclovir/valganciclovir can be administered. Here we report the results of a pair-matched analysis which compared the outcome of patients who received HSV-TK cells infusion post haplo-HSCT versus those who did not receive any cellular therapy post-transplant.Patients and Methods. The HSV-TK patients' group included 45 patients who were treated as part of 2 prospective trials with various types of high-risk hematologic malignancies. These patients were compared to patients treated with haplo-HSCT reported to the acute leukemia working party registry of the EBMT. Inclusion criteria for the pair-matched analysis encompassed haplo-HSCT transplants performed in adult patients diagnosed with AML/ALL/sAML in CR or relapse at transplantation. To equate the distribution of baseline characteristics between the HSV-TK and control group and to reduce bias in treatment effect estimation, a pair-matched analysis was performed.This analysis, in which pairs of HSV-TK and control subjects sharing similar baseline characteristics were formed, used the following parameters as pair matching factors: patient age, diagnosis (AML, ALL and sAML), disease status at HSCT (CR1, CR2, CR3 or relapse) and time from diagnosis to HSCT. The planned ratio of HSV-TK patients to control patients was one to four. Efficacy outcome measures of this pair-matched analysis were OS, LFS, NRM and relapse incidence (RI). Cumulative incidence rates of chronic GVHD were also analyzed.Results. Overall, 37 HSV-TK-treated patients matched with 140 controls (71 from PT-Cy cohort and 69 from TCD cohort transplanted between 2005 and 2013). The recommended dose and schedule of HSV-TK cells was 1x107 cells/kg given as IV infusion every 30 days for a maximum of 4 times until a circulating T-cell count higher than 100 per μL. The 1st administration should occur between day 21 to day 49 after HSCT. Baseline characteristics of the HSV-TK treated and the control patient population are summarized in the below table.OS at 1-year was significantly improved in the HSV-TK-group compared with the control group (p=0.01). The survival rates were 49% and 37% for HSV-TK- and control group, respectively. The NRM at 1-year was also improved upon treatment with HSV-TK, with 43% for the control group and 22% for the HSV-TK-group (p=0.014). A difference in favor of the HSV-TK-group could also be observed for the 1-year incidence of chronic GvHD with 25% for the control group vs 9% for the HSV-TK-group (p=0.04). The LFS and the RI were not different between the groups. Interestingly, these differences remained similar whether considering the TCD or the PT-CY subgroups). Together the data suggest that the benefit seen in OS is driven by a reduction in the NRM. A further analysis of NRM data revealed that in the control group 34 of 140 (24%) patients died due to infection and 8 of 140 (6%) succumbed due to GvHD. In the HSV-TK population 4 (11%) patients died because of infection and no patient died due to GvHD. This suggests that the reduction in NRM mortality in the HSV-TK population is caused both by a reduction in death due to infection and due to GvHD. Concerning safety, no death was attributed to HSV-TK cells. Acute GvHD resolved in all cases, and activation of the suicide gene by treatment with GCV has contributed to the control of GvHD.Conclusion. The above pair-matched analyzis confirmed the positive impact and benefit of HSV-TK cells as adjunctive treatment in haplo-HSCT with an acceptable safety pattern. [Display omitted] DisclosuresBonini:Molmed SpA: Consultancy; TxCell: Membership on an entity's Board of Directors or advisory committees. Ciceri:MolMed SpA: Consultancy.

Genome-wide association study of aggressive behaviour in chicken.

In the poultry industry, aggressive behaviour is a large animal welfare issue all over the world. To date, little is known about the underlying genetics of the aggressive behaviour. Here, we performed a genome-wide association study (GWAS) to explore the genetic mechanism associated with aggressive behaviour in chickens. The GWAS results showed that a total of 33 SNPs were associated with aggressive behaviour traits (P < 4.6E-6). rs312463697 on chromosome 4 was significantly associated with aggression (P = 2.10905E-07), and it was in the intron region of the sortilin-related VPS10 domain containing receptor 2 (SORCS2) gene. In addition, biological function analysis of the nearest 26 genes around the significant SNPs was performed with Ingenuity Pathway Analysis. An interaction network contained 17 genes was obtained and SORCS2 was involved in this network, interacted with nerve growth factor (NGF), nerve growth factor receptor (NGFR), dopa decarboxylase (L-dopa) and dopamine. After knockdown of SORCS2, the mRNA levels of NGF, L-dopa and dopamine receptor genes DRD1, DRD2, DRD3 and DRD4 were significantly decreased (P < 0.05). In summary, our data indicated that SORCS2 might play an important role in chicken aggressive behaviour through the regulation of dopaminergic pathways and NGF.

Investigating the associations between polymorphisms in the NTRK2 and NGFR genes and completed suicide in the Slovenian sample.

The most abundant neurotrophin in the mammalian brain is brain-derived neurotrophic factor (BDNF), which acts through binding to neurotrophic tyrosine kinase receptor type 2 (NTRK2) and to nerve growth factor receptor (NGFR). Our previous work showed an association of the single nucleotide polymorphism (SNP) rs6265 in the BDNF gene with completed suicide in the Slavic population. Therefore, we extended the investigation to the SNPs within NTRK2 and NGFR genes and searched for associations with the completed suicide phenotype. In 775 Caucasian individuals, namely, 486 suicide completers and 289 controls, we performed genotyping of five SNPs within the NTRK2 (rs11140714, rs1147198, rs1187323, rs10780691, and rs10868235) and six SNPs within the NGFR (rs2072446, rs7219709, rs7224806, rs734194, rs741071, and rs741072) genes. We did not find evidence for an association of the SNPs studied with the phenotype either on the single marker or on the haplotype level. To our knowledge, this is the first study that has examined SNPs in the NTRK2 and NGFR genes for associations with the completed suicide phenotype. However, our findings suggest that these SNPs may not be associated particularly with completed suicide in Slovenia, although they might have a relevant informative value as the study has been carried out on a sample from a population that has one of the highest suicide rates in the world.

Functional Ser205Leu polymorphism of the nerve growth factor receptor (NGFR) gene is associated with vagal autonomic dysregulation in humans.

Evidence indicates that reduced cardiac vagal (parasympathetic) tone, a robust cardiovascular risk factor, is a trait vulnerability marker of major depressive disorder (MDD). The Ser205/Ser205 genotype of the functional polymorphism (Ser205Leu) of the nerve growth factor receptor (NGFR), also called p75 neurotrophin receptor (p75NTR), gene is reported to increase the risk of MDD. Here, we hypothesized that the NGFR Ser205Leu polymorphism may have an effect on vagal control. A sample of 810 healthy, drug-free, unrelated Han Chinese (413 males, 397 females; mean age 35.17 ± 8.53 years) was included in the NGFR genotyping. Short-term heart rate variability (HRV) was used to assess vagus-mediated autonomic function. Potential HRV covariates, such as mood/anxiety status and serum metabolic parameters, were assessed. Homozygotes of the Ser205 allele had significantly lower high frequency power and root mean square of successive heartbeat interval differences, both HRV indices of vagal modulation, compared to Leu205 allele carriers. Even after adjusting for relevant confounders, these associations remained significant. Further stratification by sex revealed that the associations were observed only in males. Our results implicate that decreased parasympathetic activity is associated with the NGFR Ser205/Ser205 genotype in a gender-specific manner, suggesting a potential role of NGFR polymorphism in modulating cardiac autonomic function.

Association between genetic variants related to glutamatergic, dopaminergic and neurodevelopment pathways and white matter microstructure in child and adolescent patients with obsessive–compulsive disorder

BackgroundAlterations in white matter (WM) integrity observed in patients with obsessive–compulsive disorder (OCD) may be at least partly determined genetically. Neuroimaging measures of WM microstructure could serve as promising intermediate phenotypes for genetic analysis of the disorder. The objective of the present study was to explore the association between variability in genes related to the pathophysiology of OCD and altered WM microstructure previously identified in child and adolescent patients with the disease. MethodsFractional anisotropy (FA) and mean diffusivity (MD) measured by diffusion tensor imaging (DTI) and 262 single nucleotide polymorphisms (SNPs) in 35 candidate genes were assessed concomitantly in 54 child and adolescent OCD patients. ResultsSix polymorphisms located in the glutamate transporter gene (SLC1A1 rs3087879), dopamine transporter gene (SLC6A3 rs4975646), dopamine receptor D3 (DRD3 rs3773679), nerve growth factor receptor gene (NGFR rs734194 and rs2072446), and cadherin 9 gene (CDH9 rs6885387) showed significant p-values after Bonferroni correction (p≤0.00019). More specifically, the vast majority of these associations were detected with MD in the right and left anterior and posterior cerebellar lobes. LimitationsPatients were under pharmacological treatment at the time of the DTI examination. Sample size is limited. ConclusionsThe results provide the first evidence of the involvement of genetic variants related to glutamatergic, dopaminergic, and neurodevelopmental pathways in determining the WM microstructure of child and adolescent patients with OCD, which could be related to the neurobiology of the disorder.

462. Automated Manufacturing Process for the Production of Genetically Modified Lymphocytes

The MM-TK is a medicinal product constituted of T lymphocytes ex vivo genetically modified with the γ retroviral vector SFCMM-3 Mut2 #48 to express a mutated form of the Herpes Simplex Virus Thymidine kinase (HSV-TK Mut2) and a truncated form of the human Low affinity Nerve Growth Factor Receptor (ΔLNGFR) genes. It has development as adjunctive therapy in leukaemia patients or undergone bone marrow transplantation. The current manufacturing process is performed following semi-automated procedure using instrument and tubing set not marketing compliant. In order to optimize this and to standardize cellular therapeutic production for clinical trial and for the market, MolMed have strengthened an alliance with Miltenyi Biotec for tailoring of CliniMACS Prodigy platform for MM-TK DS manufacturing.A closed and automated cell process was developed using the new device with a specific software designed in order to manufacture a robust, safe and consistent product.The manufacturing process essentially consists in the stimulation of a large number of PBMCs (about 3×109 cells) isolated from an haploidentical donor, with anti-CD3 monoclonal antibody and IL-2, transduction of the stimulated cells with the SFCMM-3 Mut2 #48 retroviral vector in RetroNectin® coated bags and immunoselection of positively transduced cells using the CD271-LS Microbeads reagent (Miltenyi Biotec). Selected TK cells are then further expanded for 7 days before final formulation in freezing medium. The whole manufacturing process lasts 13 days.The following steps have been automated: cell concentration and cell washing at different manipulation steps and immunomagnetic selection of the transduced cells.Four development full-scale runs demonstrate that automated cell processing for the genetic manipulation of T lymphocytes can be performed in the closed- and automated-system designed by MolMed, reaching good results for all in-process parameters evaluated: cell recovery of 80-90%; cell viability more than 80% at each step; total process yield of 72%. The DS/DP product has been evaluated for the quality attributes as product and process related impurities (CD14% + CD16% + CD19% and residual retronectin, residual anti-CD3, residual CD271 microbeads) and potency of the product (ΔLNGFR expression; sensitivity to Ganciclovir; Vector Copy Number; immunophenotype as CD3%, CD4%, CD8%, CD45%,CD25, CD69, HLA-DR, CD95; CD45RA and CD62L). The results will support the discussion with Regulatory Autorities and permit the introduction of the new optimized closed manufacturing process in the late phase of the clinical trial and for the market.

Ginsenoside Re and Rd Enhance the Expression of Cholinergic Markers and Neuronal Differentiation in Neuro-2a Cells

In Alzheimer's disease (AD), extensive neuronal loss and a deficiency of the neurotransmitter acetylcholine (ACh) are the major characteristics during pathogenesis in the brain. In the present study, we aimed to investigate whether representative ginsenosides from ginseng can regulate choline acetyltransferase (ChAT) and vesicular acetylcholine transporter (VAChT), which are required for cholinergic neurotransmission. Our results revealed that Re and Rd induced effectively the expression of ChAT/VAChT genes in Neuro-2a cells as well as ACh elevation. Microtubule-associated protein-2 (MAP-2), nerve growth factor receptor (p75), p21, and TrkA genes and proteins were also significantly expressed. Moreover, both activated extracelullar signal-regulated protein kinase (ERK) and Akt were inhibited by K252a, a selective Trk receptor inhibitor. These findings strongly indicate that Re and Rd play an important role in neuronal differentiation and the nerve growth factor (NGF)-TrkA signaling pathway. High performance liquid chromatography analysis showed that Re and Rd administered orally were transported successfully into brain tissue and increased the level of ChAT and VAChT mRNA. The present study demonstrates that Re and Rd are selective candidates for upregulation of the expression of cholinergic markers, which may counter the symptoms and progress of AD.

Meta-analysis of genome-wide association for migraine in six population-based European cohorts

Migraine is a common neurological disorder with a genetically complex background. This paper describes a meta-analysis of genome-wide association (GWA) studies on migraine, performed by the Dutch–Icelandic migraine genetics (DICE) consortium, which brings together six population-based European migraine cohorts with a total sample size of 10 980 individuals (2446 cases and 8534 controls). A total of 32 SNPs showed marginal evidence for association at a P-value<10−5. The best result was obtained for SNP rs9908234, which had a P-value of 8.00 × 10−8. This top SNP is located in the nerve growth factor receptor (NGFR) gene. However, this SNP did not replicate in three cohorts from the Netherlands and Australia. Of the other 31 SNPs, 18 SNPs were tested in two replication cohorts, but none replicated. In addition, we explored previously identified candidate genes in the meta-analysis data set. This revealed a modest gene-based significant association between migraine and the metadherin (MTDH) gene, previously identified in the first clinic-based GWA study (GWAS) for migraine (Bonferroni-corrected gene-based P-value=0.026). This finding is consistent with the involvement of the glutamate pathway in migraine. Additional research is necessary to further confirm the involvement of glutamate.

Differential modulation of nerve growth factor receptor (p75) and cholinergic gene expression in purified p75-expressing and non-expressing basal forebrain neurons by BMP9

The synthesis of acetylcholine and its release from basal forebrain cholinergic neurons (BFCN) that innervate the cerebral cortex and hippocampus are considered essential processes for normal learning, memory and attention. We have developed a purification and cell culture method of BFCN in order to examine the regulation of their cholinergic phenotype. Cells isolated from the septal region of late embryonic mice were purified by fluorescence-activated cell sorting based on their expression of the nerve growth factor receptor (p75), a surface marker for mature BFCN. Consistent with previous reports, p75-postive (p75+) cells were enriched in choline acetyltransferase (ChAT) and the high-affinity choline transporter (ChT), as measured by reverse transcriptase PCR. In culture, these cells maintained their gene expression of p75, ChAT and ChT, while p75-negative (p75−) cells had a low expression of these genes. Incubation of the cells with BMP9 not only increased p75 and ChAT gene expression in p75− cells, but also augmented the expression of these genes in p75+ cells. Conversely, BMP9 decreased ChT gene expression in p75+ cells and had no such effect in p75− cells. Immunostaining confirmed that p75 protein expression was modulated by BMP9 in a similar way as p75 mRNA, and also revealed that only a subset of p75− cells respond to BMP9 in this manner. These data suggest that mature BFCN in culture may express their cholinergic phenotype in the absence of exogenous trophic input, but that BMP9 can further modulate this phenotype. Moreover, BMP9 induces the cholinergic phenotype in a set of basal forebrain non-cholinergic neurons or precursor cells.

Identification of immature podocyte specific antigen using retrovirus-mediated gene transfer and cell sorting

For identifying an antigen recognizable by a monoclonal antibody (mAb), the cDNA cloning method using expression cDNA library has become increasingly popular. For analysis of the mAb clone 2E3, which recognizes the cell membrane surface of immature podocytes, we developed an expression cloning strategy that identified the targeted antigen using retrovirus-mediated gene transfer and enrichment by cell sorting. In this experiment, the NIH3T3 cell line was infected by a retrovirally amplified cDNA library derived from the same fetal murine kidneys that provided the immunized antigen. Infected NIH3T3 staining for mAb clone 2E3 was concentrated by cell sorting, followed by identification of the integrated gene. The infected cells reacted with mAb were highly enriched by two rounds of cell sorting. As a result of sequencing the inserted gene from the enriched cells, we isolated the low-affinity nerve growth factor receptor (NGFR) gene. Furthermore, the NIH3T3 cell line that was enforced by recovery of NGFR cDNA reacted with mAb clone 2E3. Monoclonal antibodies recognizing cell membrane surface molecules are essential tools for immunohistochemistry and flow cytometry analysis, and the application of a combination of retrovirus-mediated gene transfer and cell sorting is beneficial for identifying the targeted antigen.

Retroviral Gene Transfer of T Cell Receptors (TCR) Specific for Minor Histocompatibility Antigens to Virus-Specific T Cells as Cellular Immunotherapy of Patients with Relapsed Hematological Malignancies after Allogeneic Stem Cell Transplantation.

Patients with hematological malignancies can be successfully treated by T cell-depleted allogeneic stem cell transplantation (alloSCT) followed by donor lymphocyte infusion (DLI). Failure of some relapsed hematological malignancies to respond to DLI is probably due to immune tolerance induced by regulatory and/or anergic T cells. We previously showed that functional T cells with redirected anti-leukemic reactivity can be generated by transfer of TCRs specific for minor histocompatibility antigens (mHags) to total peripheral blood mononuclear cells (PBMC) as well as CMV-specific T cells. By introducing TCRs into CMV or EBV specific T cells, T cells with proper memory/effector phenotypes are targeted, and due to virus persistence these T cells may show prolonged survival in vivo. The purpose of this study is to develop an efficient method for the isolation, retroviral transduction and expansion of TCR-transduced CMV- and EBV-specific T cells for cellular immunotherapy of patients with relapsed hematological malignancies after alloSCT.For clinical application, construction of single retroviral vectors coding for the α as well as β chains of mHag-specific TCRs is required. We used the MP71 retroviral vector for TCR gene transfer, since this vector contains Myeloproliferative Sarcoma Virus LTR sequences and a Mouse Embryonic Stem Cell Virus leader, which has been optimized for use in the clinic. The MP71 vector also contained a Woodchuck Hepatitis Response Element (WPRE). The WPRE, which is used as an element enhancing transgene expression at the post-transcriptional level, has recently been described to encode 60 amino acids of a protein with potential oncogenic activity. Therefore, we reconstructed the MP71 vector by introduction of a multiple cloning site (MCS) and, for safety issues, deleted the WPRE. The TCR α and β genes specific for the hematopoietic-restricted mHag HA-2 were linked by a 50-bp sequence encoding a “self-cleaving” 2A-like peptide and introduced into the MCS of the MP71 vector. Linkage of the TCR α and β genes by the 2A-like sequence allowed additional linkage of the low affinity nerve growth factor receptor (LNGFR) or human CD20 selection marker genes by an IRES sequence. The advantage of the human CD20 gene is that it can also function as suicide gene, allowing elimination of transduced cells in vivo if undesired side effects occur. Introduction of the single MP71 retroviral vector coding for the HA-2 TCR α and β chains as well as LNGFR into a TCR α- and β-deficient Jurkat T cell line led to high levels of TCR surface expression correlating with LNGFR marker gene expression. These data indicate proper cleavage and assembly of the transduced TCR α and β chains. Moreover, removal of the WPRE did not affect the surface expression level of the transduced TCR. Furthermore, CMV- and EBV-specific T cells were isolated from human individuals by the IFN-γ capture assay and subsequently transduced with a single retroviral vector coding for the HA-2-TCR α and β chains as well as LNGFR. CMV- and EBV-specific T cells from different human individuals could be successfully isolated to 60–90% purity and the TCR-transduced CMV- and EBV-specific T cells were shown to be fully functional, recognizing the viral peptides as well as the endogenously-processed HA-2 mHag.

Osteoclasts direct bystander killing of cancer cells in vitro

Cytosine deaminase (CD) catalyzes the deamination of 5-fluorocytosine (5FC) to produce the highly toxic chemotherapeutic agent 5-fluorouracil (5FU). A unique feature of the CD/5FC enzyme/prodrug system is its ability to kill adjacent cells via bystander killing. Bystander killing of cancer cells can be mediated by non-cancerous accessory cells transduced with the CD gene; one type of non-cancerous accessory cell found in primary bone cancer and breast cancer metastases to bone is the osteoclast. This manuscript determines if osteoclast precursor cells, transduced with the CD gene, can function as a gene delivery system capable of killing cancer cells. An osteoclast precursor cell line (RAW 264.7, RAW) and authentic bone marrow-derived osteoclast precursor cells were transduced with a retroviral vector containing the cytosine deaminase fusion gene (NCD) composed of the human nerve growth factor receptor and CD genes. RAW cells and bone marrow-derived osteoclast precursor cells transduced with NCD expressed NCD protein and converted 5FC to 5FU. Treatment of NCD-transduced osteoclast precursor cells with the 5FC prodrug resulted in significant killing in vitro. NCD-transduced osteoclasts were co-cultured with either DsRed2-labeled sarcoma cells (2472-DSR) or green fluorescent protein (GFP)-labeled breast cancer cells (GFP-4T1). Treatment of the NCD osteoclast/tumor cell co-cultures with 5FC resulted in bystander killing of 2472-DSR cells ( P < 0.006) and GFP-4T1 cells ( P < 0.004). These findings demonstrate that NCD-transduced osteoclasts can promote killing of cancer cells and introduce the exciting possibility for developing osteoclast-mediated, CD-based treatment of primary bone cancers and breast cancer metastases to bone.

Analysis of Distinct Tartrate-resistant Acid Phosphatase Promoter Regions in Transgenic Mice

The tartrate-resistant acid phosphatase (TRAP) is present in multiple tissues, including kidney, liver, lung, spleen, and bone. Recent study of (TRAP) gene expression has provided evidence for distinct promoters within the (TRAP) gene, suggesting that the gene has alternative, tissue-preferred mRNA transcripts. Examination of endogenous (TRAP) exon 1B and 1C mRNA transcripts revealed tissue-preferred transcript abundance with increased exon 1B transcripts detected in liver and kidney and increased exon 1C transcripts detected in bone and spleen. In this investigation, we have made transgenic mice that express a marker gene driven by two candidate promoters, designated BC and C, within the (TRAP) gene. The BC and C promoters are 2.2 and 1.6 kb, respectively, measured from the translation initiation site. Evaluation of BC transgenic lines demonstrated robust expression in multiple tissues. In contrast, significant transgene expression was not detected in C transgenic lines. Evaluation of transgene mRNAs in BC transgenic lines revealed that virtually all expression was in the form of B transcripts, suggesting that the tissue-preferred pattern of endogenous (TRAP) was not replicated in the BC transgenic line. Likewise, osteoclastogenic cultures from BC, but not C, transgenic bone marrow cells expressed the transgene following receptor activator of NFkappaB ligand/macrophage colony-stimulating factor stimulation. In conclusion, when compared with the 2.2-kb BC portion of the (TRAP) promoter region, the 1.6-kb C portion does not account for significant gene expression in vivo or in vitro; production of the bone- and spleen-preferred (TRAP) C transcript must depend on regulatory elements outside of the 2.2-kb promoter. As the majority of currently investigated transcription factors that influence transcriptional regulation of osteoclast gene expression bind within the 1.6-kb C portion of the (TRAP) promoter, it is likely that transcription binding sites outside of the 2.2-kb region will have profound effects on regulation of the gene in vivo and in vitro.